Annals of Neurology

BackgroundMutations in the E3 ubiquitin ligase Parkin (encoded by PRKN) are the most frequently known cause of recessively inherited Parkinsons disease. In addition to the loss of dopaminergic neurons, microglial activation is another pathological feature observed in Parkinsons disease. While postmortem brain samples show the end stage of the disease, neurons and glia derived from patients induced pluripotent stem cells (iPSCs) provide a model for detecting early pre-degenerative disease trajectories. However, mixed cell populations often confound these cultures, leading to heterogeneous disease phenotypes. MethodsHere, we tease apart the cell type-specific phenotypes underlying Parkin-linked Parkinsons disease by performing single-nucleus RNA sequencing in iPSC-derived co-cultures of dopaminergic neurons and microglia from PRKN mutation carriers and healthy controls. We validated our transcriptomic key findings through inflammatory cytokine profiling and live-cell calcium imaging. ResultsSingle-nucleus RNA sequencing identified seven major cell types composed of neuronal, glial, and precursor cells, with dopaminergic neurons accounting for the largest cell population. Pathway analysis revealed cell type-specific dysregulated biological processes in Parkin-deficient cells, including gene expression differences in dopaminergic neurons that control mitophagy and dopamine homeostasis, whereas microglia showed changes in calcium homeostasis and inflammatory signaling. Functional analysis verified elevated secretion of monocyte chemotactic protein 1 in PRKN-mutant co-cultures compared with controls, linking Parkin deficiency to increased microglial chemotactic signaling. Furthermore, lower intracellular calcium levels and diminished calcium release following treatment confirmed impaired calcium homeostasis in PRKN-mutant microglia. ConclusionsProfiling at the single-cell level resolved distinct cell subpopulations, enabling us to identify cell type-specific pathway disturbances underlying Parkin deficiency. This unique dataset provides a basis for understanding the impairment of individual cell types and the impact of cellular crosstalk in Parkinsons disease pathology.

BackgroundPostoperative delirium (POD) is a frequent and severe neurocognitive complication following cardiac surgery, associated with poor long-term outcomes. The underlying mechanisms are unclear, and objective biomarkers are urgently needed. MethodsWe used pre- and post-operative plasma samples from 59 patients undergoing cardiac surgery in three separate studies with rigorous delirium assessment using the Confusion Assessment Method in a case-control design. Small extracellular vesicles (sEVs) were isolated from plasma, and their miRNA cargo was profiled using RNA sequencing. Target miRNAs were validated by qRT-PCR, and digital PCR (dPCR). The functional impact of the lead candidate miRNA was investigated in vitro by assessing tau phosphorylation and cell viability in HT22 neuronal cell line. ResultsThere were no differences in sEV morphology or numbers between patients with and without POD. While three candidate miRNAs were initially validated by qRT-PCR, subsequent dPCR analysis confirmed that only the perioperative change in plasma sEV-cargo miR-330-3p expression was significantly greater in patients who developed POD (n = 20) compared with those who did not (n = 20) (5.22 copies/L plasma; 95% Confidence Interval (CI), 1.187 to 9.256; p = 0.0139). Receiver operating characteristic curve analysis for this change yielded an area under the curve of 0.745 (95% CI, 0.589 to 0.901). In vitro overexpression of miR-330-3p in a neuronal cell line significantly increased the phosphorylation of tau at Ser199 (p < 0.0001) and Ser396 (p < 0.001) and reduced cell viability (p < 0.001). ConclusionsOur findings suggest that sEV-bound miR-330-3p increases in patients with POD after cardiac surgery. In vitro results suggest a potential pathogenic role for miR-330-3p, linking a systemic signal to tau-related neuronal injury. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIThis study identifies a specific perioperative increase in small extracellular vesicle (sEV)-cargo miR-330-3p in patients with postoperative delirium (POD) following cardiac surgery. C_LIO_LIWe provide the first evidence that miR-330-3p directly induces tau hyperphosphorylation and reduces neuronal viability in vitro, establishing a potential mechanistic link between systemic sEV signaling and neurodegeneration. C_LI What Are the Clinical Implications?O_LIThe measurement of perioperative change in miR-330-3p could serve as an objective biological marker to assist in the early identification and risk stratification of patients at high risk for POD. C_LIO_LIThe identified miR-330-3p/tau pathway represents a potential new therapeutic target; future interventions aimed at inhibiting this specific miRNA might help prevent or mitigate POD-related neuronal injury. C_LIO_LIThese findings emphasize the importance of monitoring dynamic sEV-cargo changes to better understand and manage perioperative neurocognitive disorders. C_LI

BackgroundTemporal lobe epilepsy (TLE) is the most common form of focal epilepsy and is characterized by a pathological cascade of excitotoxicity that leads to neuroinflammation, progressive neuronal loss, and subsequent cognitive decline. Despite its prevalence, effective disease-modifying therapies remain lacking. Previous studies have demonstrated that the endocannabinoid system contributes to epileptic activity. In particular, inactivation of monoacylglycerol lipase (MAGL), the key rate-limiting enzyme responsible for the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG), an endogenous lipid mediator with anti-inflammatory and neuroprotective properties, suppresses seizures and reduces neuroinflammation. However, the cellular and molecular mechanisms underlying these protective effects remain unclear. MethodsTo dissect the cellular mechanisms underlying MAGL-mediated neuroprotection, we employed a kainic acid (KA)-induced status epilepticus model in mice with global, astrocyte-specific (aKO), and neuron-specific (nKO) deletion of mgll. We combined single-nucleus RNA sequencing (snRNA-seq) to map the transcriptomic landscape of glial responses with pharmacological interventions to validate key signaling pathways, as well as behavioral assays to assess functional recovery. ResultsWe demonstrated that astrocyte-specific, but not neuron-specific, mgll deletion was sufficient to attenuate seizure susceptibility and hippocampal neurodegeneration, thereby recapitulating the protective phenotype observed in global knockouts. Transcriptomic profiling revealed that astrocytic MAGL deficiency fundamentally reshaped the glial response to injury by preventing the transition to pro-inflammatory reactive astrocyte states and suppressing the activation of disease-associated microglia (DAM). Mechanistically, we identified a signaling pathway in which the neuroprotective effects of MAGL inhibition depend on cannabinoid receptor 1 (CB1) activation and are mediated by downstream peroxisome proliferator-activated receptor {gamma} (PPAR-{gamma}) signaling. Either genetic deletion of CB1 or pharmacological blockade of PPAR-{gamma} abolished the protective effects. Furthermore, aKO mice exhibited reduced neuronal loss, preserved synaptic structural integrity and protection against post-seizure cognitive deficits. ConclusionThese findings reveal astrocytic MAGL as a crucial regulatory node in the epileptic brain and demonstrated that enhancing 2-AG signaling in astrocytes orchestrates neuroprotection via CB1-PPAR-{gamma} signaling pathways, thereby attenuating neuroinflammation, preserving synaptic function, and preventing the cognitive comorbidities associated with epilepsy.

BackgroundNeuroinflammation is a common hallmark of primary tauopathies, and is associated with worse clinical outcomes over time. However, accurate prognosis in these disorders remains challenging, and current fluid biomarkers provide limited insight into the contribution of peripheral immune cells to PSP/CBS pathogenesis. Our study aims to characterise blood-based immune cell profiles in patients with progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS), and test their associations with neurodegeneration and clinical outcomes. MethodsPeripheral blood immune cells from fresh whole blood were characterized with high-dimensional mass cytometry (29 markers) in n=60 people with PSP/CBS and n=21 age- and sex-matched controls. Cell type abundance was defined as the ratio of counts for each gated population divided by total live cells. Hierarchical clustering of cell types and principal component analysis were used to derive data-driven immune clusters. Correlation network analysis and diffusion-based network propagation integrated cell counts with plasma inflammation markers to prioritise mediators of intercellular signalling. Associations between immunological markers, plasma concentrations of neurofilament light chain (NfL), cognition, and survival were assessed using regression and Cox proportional hazards models. ResultsPatients with PSP/CBS showed a global increase in covariance among immune cell populations, indicating heightened coordination within the peripheral immune network. A monocyte-driven cluster (Cluster 1) showed higher scores in PSP/CBS, reflecting impaired phenotypic transition from classical to nonclassical monocytes, and was associated with higher NfL levels, poorer cognitive performance, and worse prognosis. In contrast, a Treg-driven cluster (Cluster 2) showed lower scores in PSP/CBS, and was associated with better cognition and longer survival. Integrated multimodal networks identified a small set of immune-regulatory molecules and cytokines mediating crosstalk between Treg/Th17-like cells and monocytic populations, supporting a dysregulated Treg-monocyte axis in PSP/CBS. ConclusionsWe identified peripheral blood-based immunophenotypic profiles of individuals with PSP/CBS that are associated with neurodegeneration, cognitive decline, and survival. Dysregulated monocyte maturation and reduced Treg-related immune configurations are enriched in patients with worse outcomes, suggesting that specific peripheral immune cell subsets may serve as fluid biomarkers and potential immunotherapy targets in primary tauopathies.

BackgroundCurrent diagnostic criteria for multiple sclerosis (MS) rely on white matter lesions (WMLs), which are not specific and often occur in other disorders. Microstructural abnormalities in normal-appearing white matter (NAWM) may provide complementary information beyond focal lesions. However, the diagnostic use of NAWM in MS remains limited because a reproducible, diagnostically specific NAWM signature has not been established, and NAWM abnormalities detection typically requires quantitative MRI methods beyond routine clinical MRI protocols. MethodsIn this retrospective study, we proposed DeepMS, a deep learning model trained with both quantitative diffusion MRI (dMRI) and structural MRI (sMRI) to diagnose MS by integrating WML and NAWM features captured from routine MRI alone. Development utilized 8,450 scans from 7,703 patients (NYU Langone/ADNI). Evaluation included an internal test set (n=837) and two independent external cohorts: the Krakow cohort (Poland, n=293) and a public multi-site cohort curated from 15 datasets (n=1,756). We compared DeepMS against 2024 McDonald criteria biomarkers (Dissemination in Time [DIT], Dissemination in Space [DIS], Central Vein Sign [CVS], and Paramagnetic Rim Lesion [PRL]) in a multireader study (n=308). To validate the models use of NAWM, we performed lesion-masking experiments (n=550), comparing performance after removal of focal lesions. FindingsDeepMS achieved robust AUCs in the internal (0{middle dot}968 [95% CI 0{middle dot}946-0{middle dot}987]), Krakow (0{middle dot}940 [0{middle dot}898-0{middle dot}974]), and public external (0{middle dot}974 [0{middle dot}966-0{middle dot}982]) cohorts. In the multireader study, DeepMS outperformed established biomarkers: at matched sensitivity (92{middle dot}9%), DeepMS achieved higher specificity than DIS (89{middle dot}0% vs 78{middle dot}5%; p=0{middle dot}0061); at matched specificity (92{middle dot}8%), DeepMS achieved higher sensitivity than CVS (88{middle dot}2% vs 52{middle dot}0%; p<0{middle dot}0001). Furthermore, DeepMS retained diagnostic capability after WML masking (AUC 0{middle dot}959 to 0{middle dot}881) compared to the model trained with only sMRI (0{middle dot}895 to 0{middle dot}764). InterpretationOur findings suggest it is feasible for deep learning models to leverage NAWM-related information directly from routine sMRI. Integrating these features could help MS diagnosis in patients with ambiguous white matter abnormalities. FundingNational Institute of Neurological Disorders and Stroke, the National Institute of Biomedical Imaging and Bioengineering, and the Irma T. Hirschl Trust.

Background: Acute necrotizing encephalopathy (ANE) is a rare and severe neurologic complication of viral infection in children, thought to result from a hyperacute cytokine storm causing blood-brain barrier disruption and central nervous system injury. Despite characteristic clinical and radiologic features, ANE remains poorly understood at the molecular level, with no validated biomarkers or targeted therapies. We aimed to determine whether genetic predisposition to ANE due to RANBP2 variants is associated with a distinct immunologic signature. Methods: We conducted a prospective biological study of familial ANE (ANE1, NCT06731790). We included 23 heterozygous carriers of the RANBP2 c.1754C>T (p.Thr585Met) variant from 10 families, and 28 noncarriers (median age, 40 years [range, 4-72]). Soluble immune mediators, transcriptomic analyses, multiparameter flow cytometry, and cellular imaging were analysed in peripheral blood mononuclear cells (PBMCs) and monocytes. Baseline and resiquimod stimulated immune responses were analysed within the same statistical model, with genetic status as the primary predictor. Findings: The RANBP2 Thr585Met mutation was associated with a dysregulated inflammatory phenotype characterized by reduced basal mediator production and exaggerated TNF- responses following stimulation (estimated difference, +2,098 pg/mL; 95% CI, 1,121 to 3,076; P=0.0001). Transcriptomic and flow cytometry analyses showed broad reprogramming of myeloid cells with enrichment of CXCR3-high CD14-high subsets. Expansion of these populations was associated with increased long-term disease burden. The RANBP2 variant was the only independent factor associated this inflammatory phenotype. Interpretation: RANBP2-associated ANE is characterised by a distinct immunological signature that can inform disease stratification and support the development of targeted immunotherapeutic approaches.

ImportanceTracking and predicting seizure frequency in patients with epilepsy is important for prognostication and therapy management. Interictal spikes have been proposed as a biomarker of seizure burden, but their association with seizure frequency has not been well quantified across epilepsy subtypes. ObjectiveTo measure the association between spike rate and seizure frequency and how this varies by epilepsy subtype. Design, Setting and ParticipantsWe studied 3,614 consecutive routine outpatient EEGs from 3,245 patients with epilepsy. A validated automated detector (SpikeNet2) estimated spike frequency. Validated large language models performed natural language processing on outpatient clinic notes to extract seizure frequency and epilepsy subtype. Main Outcomes and MeasuresSpearman correlation between spike frequency (spikes/hour) and seizure frequency (seizures/month) for all patients with epilepsy and for patients with generalized epilepsy, temporal lobe epilepsy, and frontal lobe epilepsy. ResultsOverall, spike frequency was modestly associated with seizure frequency (N = 3,245, {rho} = 0.11, p < 0.001). Significant positive associations were observed in generalized epilepsy (N = 625, {rho} = 0.23, Bonferroni-adjusted p < 0.001) and temporal lobe epilepsy (N = 834, {rho} = 0.12, p = 0.0013), but not in frontal lobe epilepsy (N = 263, {rho} = 0.11, p = 0.22). Conclusions and RelevanceIn this large outpatient cohort, higher interictal spike rates on routine EEG were associated with higher seizure frequencies, with the strongest relationship observed in generalized epilepsy. These associations support interictal spike rate as a quantitative EEG marker of seizure burden. Spike rate may have clinical utility for risk stratification at diagnosis and for monitoring longitudinal changes in seizure burden in response to therapy.

ObjectiveLevetiracetam is commonly prescribed for seizure prophylaxis after acute ischemic stroke (AIS) and often continued beyond discharge. While its short-term effectiveness for preventing post-stroke seizures is established, it is unclear whether prolonged use improves survival, particularly in older adults. We estimated the effect of continued levetiracetam use on 90-day mortality among Medicare beneficiaries after AIS. MethodsUsing Traditional Medicare claims data (2008-2021), we identified beneficiaries aged [&ge;]66 years hospitalized for AIS who initiated outpatient levetiracetam within 90 days of discharge. After one month of continued post-stroke use of levetiracetam (start of follow-up), we compared 90-day mortality between patients with a new levetiracetam dispensation within a 14-day grace period post-follow up and those without one. We performed cloning, censoring and weighting to address immortal time bias and estimated standardized mortality risks, risk differences, and 95% confidence intervals (CI). ResultsAmong 3,212 eligible beneficiaries, 1,779 (55.4%) received a new levetiracetam dispensation within the 14-day grace period. Median age was 76 years (IQR 70-83); 57.8% were female. After adjustment for demographics, hospitalization characteristics, timing of initiation, and comorbidities, continued use was associated with lower 90-day mortality than discontinuation (53 vs 62 deaths per 1,000; risk difference -9 per 1,000; 95% CI: (-12,-5)). The reduction was observed primarily among patients aged [&ge;]75 years. SignificanceAmong older Medicare beneficiaries who initiated levetiracetam after AIS, continued outpatient use was associated with modestly lower 90-day mortality, particularly in those aged [&ge;]75 years. These findings suggest potential benefits of levetiracetam continuation beyond the immediate post-stroke period.

BackgroundEpidemiological studies show an inverse association between cigarette smoking and Parkinsons disease (PD), suggesting a potential protective effect of smoking on PD incidence, despite the well-established and overwhelming harms of smoking to human health. We integrated genomic and proteomic approaches to investigate the causality and molecular basis of this potential relationship. MethodsWe analyzed summary statistics from genome-wide association studies (GWAS) of smoking initiation (SmkInit), smoking intensity, and PD. Two-sample Mendelian randomization (MR) tested whether genetic liability to smoking behaviors causally influences PD risk. Shared genomic architecture was quantified using MiXeR, and conjunctional false discovery rate (conjFDR) analysis identified loci jointly associated with smoking and PD, which were then mapped to genes and tested for tissue enrichment. To identify mediating proteins, we integrated dorsolateral prefrontal cortex proteomic data with GWAS using proteome-wide association studies (PWAS), summary-based MR, heterogeneity in dependent instruments testing, and colocalization. Finally, the druggability of convergent genes was evaluated. ResultsMR analyses indicated a protective effect of genetic liability to SmkInit on PD risk (OR = 0.78, 95% CI: 0.67-0.91, P = 1.5 x 10-3), which was consistent across sensitivity analyses and not suggestive of directional pleiotropy. However, no significant effect of genetic liability to cigarettes per day (CigDay) on PD risk was found. MiXeR revealed modest polygenic overlap between SmkInit and PD (13.9%; genetic correlation rg = -0.16) and between CigDay and PD (22.9%; rg = -0.09). ConjFDR identified 95 shared loci for SmkInit-PD and 26 for CigDay-PD. SmkInit-PD loci mapped to genes involved in neurotrophic signaling, synaptic organization, microglial modulation, and mitochondrial stress responses, with expression enriched in substantia nigra, basal ganglia, and interconnected cortical regions. PWAS identified 11 proteins shared by PD and SmkInit and 5 shared with CigDay, several of which (AKT3, MAPT, RIT2, EXD2, and PPP3CC) were supported by both genomic and proteomic analyses. Druggability assessment highlighted six proteins with existing pharmacologic modulation potential, spanning neurotrophic, microglial, proteostatic, and ion-channel pathways. ConclusionsGenetic liability to smoking initiation appears to confer modest protection against PD. Integrative genomic and proteomic evidence converges on neurotrophic, synaptic, microglial, and mitochondrial pathways as shared mechanisms, identifying biologically coherent potential therapeutic targets for advancing smoke-free neuroprotective strategies in PD.

BackgroundAnti-seizure medications (ASMs) are widely used in neonatal intensive care, but there is limited evidence for their safety and long-term outcomes. Phenobarbital is the only ASM generally recommended for use in neonates, but it has been linked with adverse effects in infants. Other anti-seizure medications, such as fosphenytoin, levetiracetam, and midazolam are used off-label in this population. MethodsWe performed a retrospective observational study of 18,548 infants in intensive care at an academic medical center, examining links between neonatal ASM exposure and neurological outcomes over the follow-up period of median 4{middle dot}5 years (IQR 1{middle dot}6 - 9{middle dot}2 years). The real-world clinical data included comprehensive maternal, perinatal, and medication data. The outcomes of interest were cerebral palsy, epilepsy, intellectual disability, and visual impairment. Multivariable cause-specific Cox models were used to estimate hazard ratios (HRs) for phenobarbital, levetiracetam, midazolam, and fosphenytoin exposure. Models were adjusted for major perinatal confounders, including gestational age, birth weight, mode of delivery, intraventricular hemorrhage, hypoxic-ischemic encephalopathy, and stroke. FindingsExposure to the median cumulative dose of phenobarbital was associated with increased HR for epilepsy (HR 1{middle dot}35; 95% CI, 1{middle dot}11-1{middle dot}62, p = 0{middle dot}002) visual impairment (HR 1{middle dot}20; 95% CI, 0{middle dot}99-1{middle dot}45, p = 0{middle dot}06), and intellectual disability (HR 1{middle dot}18; 95% CI, 0{middle dot}99-1{middle dot}41, p = 0{middle dot}06). In contrast, levetiracetam was associated with smaller risk increases for cerebral palsy (HR 1{middle dot}13; 95% CI, 1{middle dot}03-1{middle dot}23, p = 0{middle dot}006, epilepsy (HR 1{middle dot}14; 95% CI 1{middle dot}05-1{middle dot}24, p = 0{middle dot}002 and visual impairment (HR 1{middle dot}18; 95% CI 1{middle dot}11-1{middle dot}26, p <0{middle dot}0001). Midazolam exposure was associated with slightly increased risk of intellectual disability (HR 1{middle dot}09, 95% CI, 1{middle dot}02- 1{middle dot}16). Results for fosphenytoin were statistically not significant. We did not find evidence of a dose-dependent effect of phenobarbital, but increased maximum phenobarbital blood concentration were associated with elevated hazard ratios for cerebral palsy (HR 1{middle dot}48; 95% CI, 1{middle dot}07-2{middle dot}06, p = 0{middle dot}02 for 50 {micro}mol/l increase) and epilepsy (HR 1{middle dot}64; 95% CI, 1{middle dot}14-2{middle dot}35, p = 0{middle dot}007 for 50 {micro}mol/l increase). InterpretationThe results align with previous findings linking phenobarbital to neurodevelopmental harm and emphasize the need for its cautious use in neonates. Levetiracetam had more favorable safety profile. These findings highlight the potential of real-world data to inform evidence-based neonatal pharmacotherapy when randomized trials are impractical. FundingThe Foundation for Pediatric Research (Finland), the Association of Friends of the University Childrens Hospitals (Lastenklinikoiden Kummit ry), and internal institutional funding.

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motor neuron loss accompanied by neuroinflammation, oxidative stress, and impaired proteostasis, with pathological aggregation of TDP-43 as its defining hallmark. Microglia are recognized as central contributors to ALS pathogenesis, yet the mechanisms underlying their intrinsic dysfunction in the context of TDP-43 remain insufficiently characterized. MethodsWe characterized microglia-like cells (iMGLs) differentiated from isogenic TDP-43 M337V human induced pluripotent stem cell lines. We performed integrated functional assays, transcriptomic profiling and drug repurposing analysis to systematically compare mutant and control iMGLs. To assess therapeutic potential, we evaluated the effects of ROPI, a dopamine D2 receptor agonist previously advanced to clinical trials for ALS, on disease-relevant phenotypes in TDP-43 iMGLs. ResultsTDP-43M337V/M337V iMGLs presented ALS-associated abnormalities, including cytoplasmic TDP-43 accumulation with impaired phagocytosis and elevated oxidative stress, impaired autophagy and mitophagy, altered cytokine profiles, and reduced ferritin levels. Building on our previous identification of ropinirole hydrochloride as neuroprotective for ALS motor neurons, we now show that it confers therapeutic benefits in mitigating microglial pathology. ROPI treatment significantly reduced oxidative stress and caspase-3/7 activity and partially restored cytokine homeostasis in TDP-43M337V/M337V iMGLs, independent of autophagy/mitophagy modulation. Transcriptomic profiling revealed that ropinirole modulated disease-associated gene expression signatures involving protein folding, extracellular matrix organization, and oxidative stress responses. Furthermore, connectivity map-based analysis prioritized PI3K-Akt-mTOR inhibitors as candidates for reversing TDP-43 iMGL signatures, and ropinirole was found to modulate mTOR signaling. These converging lines of evidence support a mechanistic role for ropinirole in restoring microglial homeostasis via PI3K-mTOR pathway regulation. Taken together, our findings position ropinirole as a promising candidate dual action therapeutic candidate capable of targeting both neuronal and microglial dysfunction in ALS, suggesting broader applicability of ropinirole in modulating neuroinflammatory cascades across other neurodegenerative conditions. ConclusionRopinirole broadens ALS therapy from motor neurons to microglia, underscoring its promise for integrated and clinically meaningful treatment strategies.

Focal cortical dysplasias (FCDs) are one of the most common structural causes of drug-resistant epilepsy in children but are frequently subtle and difficult to detect on conventional MRI. Many automated lesion detection methods have therefore been proposed to support neuroradiological assessment. In this study, we externally validated two recently developed deep-learning approaches for FCD detection, MELD Graph and 3D-nnUNet, in a pediatric cohort. In this retrospective single-center study, brain MRI scans of 71 children evaluated for epilepsy were analyzed, including 35 MRI-positive patients with suspected FCD and 36 MRI-negative cases based on the primary radiology reports. Both models were applied to standard 3D T1-weighted and 3D FLAIR images. Detected lesions were reviewed by an experienced pediatric neuroradiologist and classified as true positive, false positive, or false negative. Clinical semiology and EEG findings were additionally evaluated for cases with false-positive detections. At the lesion level, MELD Graph achieved a precision of 0.85 and recall of 0.52, while 3D-nnUNet achieved a precision of 0.91 and recall of 0.48. In the MRI-negative patients, MELD Graph produced more false-positive detections than 3D-nnUNet (0.53 vs. 0.14 false-positive lesions per patient). At the patient level, MELD Graph showed slightly higher sensitivity than 3D-nnUNet (0.63 vs. 0.54), whereas 3D-nnUNet demonstrated markedly higher specificity (0.86 vs. 0.56). Improved FLAIR image quality was associated with trends toward improved model performance. Both models demonstrated high precision but moderate sensitivity, indicating that they are valuable decision-support tools but cannot replace expert neuroradiological evaluation. Optimized MRI acquisition protocols are needed to further improve automated lesion detection in pediatric epilepsy.

BackgroundPrognosis and therapeutic management in Parkinsons disease remain challenging due to the diseases heterogeneous progression and symptom presentation and lack of reliable biomarkers to predict individual disease trajectories. ObjectiveTo determine whether baseline blood transcriptomes, analyzed through biologically defined pathway gene sets, contain signatures that distinguish distinct motor and non-motor progression trajectories in Parkinsons disease. MethodsUsing data from the Parkinsons Progression Markers Initiative cohort, we developed a pathway-based computational framework to derive individualized molecular severity scores from baseline blood transcriptomic profiles by integrating pathway-level gene expression with longitudinal clinical data. Severity indices for motor and non-motor features established domain-specific progression trajectories of sporadic Parkinsons disease. Machine learning models were trained to predict patient trajectory membership from baseline transcriptomics. Findings were validated in genetic subcohorts and externally in the Parkinsons Disease Biomarkers Program cohort. ResultsMolecular severity scores were associated with key clinical features. Analysis of score changes revealed two non-motor and two motor progression groups, each characterized by specific gene signatures (20 genes for non-motor; 121 for motor). From baseline transcriptomic data, we accurately predicted an individuals trajectory group (0.87 for motor progression). The framework demonstrated high generalizability across independent and genetic cohorts, producing clinically coherent profiles. ConclusionsOur analysis reveals that baseline blood transcriptomic profiles delineate motor and non-motor progression trajectories in sporadic Parkinsons disease. The results are consistent with prior findings and may contribute to the identification of novel biomarkers, thereby informing and potentially optimizing the design of clinical trials aimed at modifying disease progression.

BackgroundAphasia commonly occurs after left-hemispheric stroke, yet substantial inter-individual variability in language outcomes remains insufficiently explained by established clinical systems neuroscience concepts. Emerging evidence suggests that the integrity of specific neurotransmitter systems may influence functional outcomes after stroke. This study examined whether the damage to neurotransmitter-related structural networks is associated with post-stroke language impairment. MethodsData of 270 patients with left-hemispheric stroke from two openly available cohorts were analyzed: the acute Washington Stroke Cohort and the chronic Aphasia Recovery Cohort. Neurotransmitter-related network damage was quantified by embedding individual stroke lesion masks into normative connectomes weighted by PET-derived density maps of 16 neurotransmitter receptors and transporters. Partial least squares (PLS) regression identified informative predictors of language functioning, followed by linear regression analyses adjusted for age, sex, lesion volume, and time post-stroke. ResultsAcross both cohorts, PLS analyses converged on a neurochemical profile in which damage to networks related to serotonergic (5-HT1a, 5-HT2a) and dopaminergic (D1) receptor distributions showed the strongest associations with poorer language performance. Damage to the 5-HT1a and D1-related networks remained significant in fully adjusted models, leading to substantially improved model fit. ConclusionThe disruption of large-scale serotonergic (5-HT1a) and dopaminergic (D1) brain networks is associated with language impairment in acute and chronic stroke. Neurotransmitter-related network damage explained additional variability in language performance beyond clinical variables and lesion burden. This work adds a neurochemically informed network perspective to aphasia research and may pave the way for future biological patient stratification to support targeted rehabilitation strategies, such as pharmacological interventions.

ImportanceAccurate diagnosis of neurodegenerative movement disorders is challenging because of a lack of in vivo biomarkers, overlapping clinical features and a delay in the emergence of pathognomonic features. ObjectiveTo evaluate clinicopathological correlation, diagnostic accuracy, genetic association with pathology, and ancestry-related differences in a multi-ancestry brain bank cohort. DesignMulticentre retrospective autopsy cohort study on donors enrolled between 1985 - 2024. Setting11 academic brain banks in the UK, US and Australia ParticipantsBrain donors identified from participating brain banks with available brain tissue and a clinical diagnosis of Parkinsons disease, Parkinsons disease dementia, dementia with Lewy bodies, progressive supranuclear palsy, corticobasal syndrome, multiple system atrophy, or neurologically normal controls. ExposureGenetic variant carrier status and clinical diagnostic category. Main outcomeClinical diagnostic accuracy; Lewy body and Alzheimers disease pathology burden; survival; association with genetic variants and genetically inferred ancestry. ResultsWe studied 3,353 brain donors (1281 [38.2%] female, mean [SD] age at death, 76.8 [10.6] years). Misdiagnosis rates for movement disorders ranged approximately from 10% - 20%. Clinical diagnoses of dementia with parkinsonism (PDD/DLB) were more strongly associated with Lewy body pathology than Parkinsons disease without dementia (OR = 1{middle dot}96, 95% CI = 1{middle dot}30 - 3{middle dot}04, p = 7{middle dot}2e-04). Lewy pathology was identified in 4% of neurologically normal controls. Alzheimers disease co-pathology was present in 40% of cases with Lewy body disease. GBA1 variant carriers exhibited greater Lewy body burden compared with noncarriers (OR = 1{middle dot}94, 95% CI = 1{middle dot}24 - 3{middle dot}03, p = 0{middle dot}01) or LRRK2 carriers (OR = 7{middle dot}44, 95% CI = 2{middle dot}16 - 25{middle dot}64, p = 0{middle dot}01). Pathological diagnoses differed by ancestry, with South Asian donors more likely to have progressive supranuclear palsy pathology and Ashkenazi Jewish donors more likely to have Lewy body disease (p < 0.0001), independent of GBA1 and LRRK2 mutation status. Conclusion and RelevanceOur findings highlight the value of integrating genetic and pathological data to improve diagnostic accuracy. The high prevalence of Alzheimers disease co-pathology and ancestry-related differences in pathology point to the need for biologically informed diagnostic tools. These results support the integration of genetically and pathologically stratified approaches, correlating pathology with in vivo biomarkers, for future therapeutic trials. FundingMedical Research Council, Global Parkinsons Genetic Program/Aligning Science Across Parkinsons Key PointsO_ST_ABSQuestionC_ST_ABSHow do genetic variants and neuropathology influence clinical features and diagnostic accuracy in movement disorders? FindingsIn this multi-ancestry brain bank series including over 3000 individuals, clinical misdiagnosis was common. Dementia with parkinsonism was more strongly associated with Lewy body (LB) pathology than Parkinsons disease without dementia, and Alzheimers disease co-pathology was frequent. Genetic variation was associated with pathological differences. GBA1 carriers had greater LB burden, while LRRK2 pathogenic variant carriers had a lower LB burden and longer survival. Pathological diagnosis differed by ancestry. MeaningIntegrating genetics and neuropathology may improve diagnosis and support pathology-informed therapeutic trials.

Background: Sleep wake and circadian disturbances are increasingly recognised in people living with amyotrophic lateral sclerosis (plwALS), but endogenous circadian phase timing and its prognostic significance in early disease remain unclear. We assessed whether salivary dim-light melatonin onset (DLMO), an objective marker of central circadian phase, is altered in early plwALS and whether it provides prognostic information. Methods: In this prospective longitudinal observational study, plwALS within 18 months of symptom onset underwent home-based salivary melatonin sampling under dim light conditions at six predefined time points around habitual sleep onset (HSO). Melatonin profiles were modeled using cubic smoothing splines, and DLMO was defined as the first time the fitted curve reached 3 pg/mL. Clinical, respiratory, and sleep assessments were collected at baseline (T0) and after 6 months (T6); a subgroup repeated saliva sampling at T6. Age and sex matched controls underwent melatonin profiling. Associations with disease progression, incident respiratory symptoms, and survival/tracheostomy were examined using regressions and survival analyses. Results: Fifty plwALS were enrolled. Compared with controls, plwALS showed an earlier DLMO (20:24 vs 20:58; p=0.028) despite similar HSO and chronotype. Within ALS cohort, a later baseline DLMO correlated with worse functional/motor status, faster progression of disease, incident dyspnea/orthopnea by T6 (adjusted OR 3.02; p=0.017), and poorer survival/tracheostomy-free outcome. In re-sampled subgroup (n=28), DLMO and other melatonin-derived metrics did not change over 6 months. Conclusions: Circadian phase alterations are detectable in early ALS. Baseline DLMO may represent a non-invasive prognostic biomarker for progression, respiratory symptom emergence and survival, warranting validation in larger multicentre cohorts.

IntroductionNeurodegenerative dementia syndromes are severely debilitating, progressive and increasing in incidence with an ageing population. A treatable differential diagnosis to neurodegenerative dementia is autoimmune encephalitis (AE), but AE patients are often misdiagnosed, delaying treatment. Previous work in the Netherlands has shown that 0.8% of patients with suspected neurodegenerative dementia suffer from AE. In Sweden, there is considerable variability in the prevalence of AE, possibly indicating under-diagnosis. We hypothesized that some Swedish individuals seeking care for memory impairment might suffer from an undetected AE and that these would show aberrances in available markers of neuroinflammation. MethodsWe retrospectively screened frozen sera from 1041 individuals seen between 2019 and 2023 at the Karolinska University hospital memory clinics in Stockholm for autoantibodies to contactin-associated protein-like 2 (CASPR2), leucine-rich glioma-inactivated 1 (LGI1), gamma-aminobutyric acid receptor B (GABABR), the n-methyl-d-aspartate receptor (NMDA-R) and Immunoglobulin superfamily containing LAMP, OBCAM, and Neurotrimin family member 5 (IgLON5) using live cell-based assays (CBAs) and scored them by microscopy. Serum and CSF from suspected positive patient samples were re-tested and titrated by live CBA, commercial fixed CBAs and tissue based assays. Results8 of the 1021 individuals, or 0.8% of the cohort, tested positive in at least three different tests for antibodies to CASPR2 (n=3), GABABR (n=2), LGI1 (n=1) and NMDAR (n=2). Seven of these patients had not been previously diagnosed with AE. Apart from two CASPR2-antibody positive patients showing neuropathic pain and seizures and neuromyotonia, respectively, the patients lacked clinical signs of encephalitis aside from memory impairment and affect lability. The antibody-positive patients did not differ significantly from autoantibody-negative patients in any available clinical parameter. None showed signs of inflammation on brain magnetic resonance tomography, and 2/7 lacked any sign of neuroinflammation in the CSF with available tests, which is commonly seen in later-onset AE. ConclusionOur work identifies undiagnosed AE patients with subtle symptomatology among Swedish memory clinic visitors, that cannot be sensitively separated from antibody-negative patients with current diagnostic tests. Our results suggest the need for the introduction of more sensitive markers of neuroinflammation to the memory clinic to identify and treat individuals with AE among sufferers of memory impairment.

BackgroundALS is increasingly recognized as a biologically heterogeneous disease in which several molecular and pathological mechanisms converge on a similar clinical phenotype. One of these molecular markers is ferritin accumulation which is observed in a subset of ALS cases and has been shown to directly correlate with TDP-43 pathology in some brain regions. Additionally, TDP-43 proteinopathy is observed outside of ALS which may complicate the interpretation of case vs control approaches to target discovery. Here, we propose a pathology-stratified approach to empower targeted theranostics. We hypothesised that biologically distinct ALS subtypes may be defined by specific metabolic dysfunction linked to brain-accumulated ferritin and TDP-43 pathology. MethodsPost-mortem primary motor cortex tissue from 15 ALS cases and 20 age- and sex-matched controls was stratified, using immunohistochemistry, by single- or co-occurrence of ferritin accumulation, and pathological TDP-43. Untargeted metabolomics (>1,000 metabolites) was performed, and samples were stratified into dual positive (ferritin and TDP-43), single positive (either), or negative. Group-discriminating metabolites were identified using partial least squares discriminant analysis. ResultsDual ferritin and TDP-43 pathology reflected a distinct metabolomic profile, separable from single-pathology states. This dual positive metabolic signature was characterised by disruption of lysophospholipid, lysoplasmalogen, and fatty acid metabolism, consistent with impaired membrane and energy homeostasis. In contrast, pathological TDP-43 presence without ferritin, was characterised metabolically by significant depletion of secondary bile acids and increase in glycosylation markers, whilst ferritin accumulation alone reflected significant increase in oxidative stress and depletion of lipid peroxidation inhibition markers. The dual positive state suggests failure of compensatory metabolic responses present in single-pathology conditions. ConclusionsFerritin accumulation and TDP-43 pathology define biologically distinct subtypes associated with ALS with divergent metabolic vulnerabilities. The metabolic signature associated with dual pathology provides a mechanistic correlate to MRI-visible ferritin accumulated iron, supporting paired non-invasive biomarker and target discovery for pathology-dependent patient stratification. These findings argue for pathway-targeted, subtype-specific therapeutic strategies and highlight the necessity of precision medicine approaches in ALS. Short abstractAmyotrophic lateral sclerosis (ALS) exhibits profound molecular heterogeneity that is not captured by current clinical classifications. Additionally, TDP-43 proteinopathy is observed outside of ALS which may complicate the interpretation of case vs control approaches to target discovery. Here, we propose a pathology-stratified approach to therapeutic target discovery, identifying convergent iron dysregulation and TDP-43 pathology with specific metabolic consequences. Post-mortem primary motor cortex tissue from 15 ALS cases and 20 controls was investigated for ferritin, and pathological TDP-43 using RNA aptamer-based immunostaining. Untargeted metabolomics (>1,000 metabolites) was performed with stratification into dual positive, single positive, or negative groups, followed by partial least squares discriminant analysis. Dual ferritin and TDP-43 pathology produced a distinct metabolic state characterised by disruption of lysophospholipid, lysoplasmalogen, and fatty acid metabolism, indicating impaired membrane integrity and energy homeostasis. In contrast, single positive states engaged divergent compensatory pathways involving bile acid metabolism, glycosylation, or oxidative stress regulation. Ferritin-TDP-43 convergence defines a metabolically decompensated ALS subtype corresponding to MRI signatures, providing a mechanistic basis for imaging-guided, pathology-dependent patient stratification and targeted intervention. Key FindingsO_LIMetabolically distinct subtypes were defined by the presence or absence of ferritin-associated iron accumulation and TDP-43 pathology in the primary motor cortex. C_LIO_LIConcurrent ferritin and TDP-43 pathology produce a unique, metabolically decompensated state characterised by disrupted lipid, membrane, and energy metabolism, distinct from either pathology alone. C_LIO_LISingle positive states engage divergent compensatory metabolic pathways, which are lost when ferritin and TDP-43 co-occur. C_LIO_LIThe metabolic signature of dual positivity provides a mechanistic correlate to the MRI-visible motor band sign. C_LIO_LIThese findings support the use of pathology-based stratification of ALS patients and a foundation for pathway-targeted, precision therapeutic approaches. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/711539v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@69d482org.highwire.dtl.DTLVardef@1fee3a4org.highwire.dtl.DTLVardef@1135017org.highwire.dtl.DTLVardef@ef3f96_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundAmyotrophic lateral sclerosis (ALS) is clinically heterogeneous, and genetic modifiers may drive molecular endophenotypes without obvious clinical stratification. The apolipoprotein E {varepsilon}4 (APOE {varepsilon}4) allele is a major Alzheimers disease risk allele, but its biological impact in ALS remains unclear. MethodsUsing the Answer ALS cohort, longitudinal motor, cognitive, and neuropsychiatric measures were modelled using mixed-effects approaches. Patient induced pluripotent stem cell-derived motor neuron multiomics (chromatin accessibility, transcriptomics, and proteomics) were analysed using supervised machine learning. Plasma SomaScan profiling was used to derive an APOE {varepsilon}4-associated protein signature and to test its stability across serial visits, biological pathway enrichment, and associations with clinical progression. ResultsAPOE {varepsilon}4 carriage was not associated with baseline severity or rate of functional decline and showed no consistent effects on cognitive or neuropsychiatric trajectories. Motor neuron multiomic profiles similarly demonstrated no reproducible APOE {varepsilon}4 signal and did not reliably classify genotype. In contrast, plasma proteomics identified an APOE {varepsilon}4 protein signature that classified {varepsilon}4 status with high accuracy in ALS (AUC 0.98) and non-ALS motor neuron disease (AUC 0.86) and was enriched for immune and inflammatory biology. This systemic signature was highly stable across repeated sampling, indicating a persistent genotype-associated state. Within this plasma endophenotype, a small set of proteins tracked functional decline and a composite score stratified fast versus slow progression. Baseline composite scores were elevated in APOE {varepsilon}4 carriers in both ALS and neurologically unimpaired controls, consistent with a stable systemic shift detectable beyond overt disease status. ConclusionsAPOE {varepsilon}4 defines a persistent, immune-enriched systemic proteomic endophenotype in ALS that is not captured by clinical trajectories or motor neuron-only profiling yet relates to disease progression. Plasma-based, genotype-informed endophenotyping offers a translational pathway for biomarker stratification and therapeutic prioritisation in ALS and potentially other heterogeneous neurodegenerative disorders.

Background: Peri-lead edema (PLE) occurs in up to 15% of Deep Brain Stimulation (DBS) cases, can cause morbidity, and its etiology remains unknown. We hypothesized that PLE represents a secondary brain injury modulated by hypoxemia, and that patients with obstructive sleep apnea (OSA) are at elevated risk. Methods: We conducted a retrospective case-control study of 121 Parkinson's disease (PD) patients undergoing DBS at a single center (2019-2024). PLE severity was quantified by CT volumetric segmentation and Hounsfield unit (HU) measures. Perioperative SpO2 and PaO2 were recorded. Polysomnography (PSG) was available in 26 patients; and the REM Sleep Behavior Disorder Screening Questionnaire (RBDSQ) was administered retrospectively. Results: Symptomatic PLE occurred in 12 patients (9.9%), with onset at 3.5 (2-9) days postoperatively. PLE patients had higher body mass index (p = 0.022) and higher OSA prevalence (75% vs. 30%; p = 0.002). Perioperative SpO2 was lower in the PLE group in both the operating room and post-anesthesia care unit (PACU; p < 0.05); PaO2 was lower in the PACU (p = 0.037). In the PSG subgroup, REM Sleep Behavior Disorder (RBD) incidence was lower in PLE patients (20% vs. 60%; unadjusted p = 0.048), and PLE severity correlated significantly with sleep-related hypoxemia and respiratory indices. RBDSQ scores were positively associated with edema density (normalized HU: rho = 0.86, p = 0.024). Conclusions: OSA and perioperative hypoxemia are associated with symptomatic PLE following DBS, while RBD appears protective. Preoperative sleep evaluation and optimized perioperative airway management warrant prospective investigation as PLE prevention strategies.